The Application of Ozonated Water Rearranges the Vitis Vinifera L. Leaf and Berry Transcriptomes Eliciting Defence and Antioxida

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OPEN The application of ozonated water rearranges the Vitis vinifera L. leaf and berry transcriptomes eliciting defence and antioxidant responses Ana Campayo1,2,5, Stefania Savoi3,5, Charles Romieu3, Alberto José López‑Jiménez4, Kortes Serrano de la Hoz2, M. Rosario Salinas1, Laurent Torregrosa3* & Gonzalo L. Alonso1

Ozonated water has become an innovative, environmentally friendly tool for controlling the development of fungal diseases in the vineyard or during grape postharvest conservation. However, little information is currently available on the efects of ozonated water sprayings on the grapevine physiology and metabolism. Using the microvine model, we studied the transcriptomic response of leaf and fruit organs to this treatment. The response to ozone was observed to be organ and developmental stage-dependent, with a decrease of the number of DEGs (diferentially expressed genes) in the fruit from the onset of ripening to later stages. The most highly up-regulated gene families were heat-shock proteins and chaperones. Other up-regulated genes were involved in oxidative stress homeostasis such as those of the ascorbate–glutathione cycle and glutathione S-transferases. In contrast, genes related to cell wall development and secondary metabolites (carotenoids, terpenoids, phenylpropanoids / favonoids) were generally down-regulated after ozone treatment, mainly in the early stage of fruit ripening. This down-regulation may indicate a possible carbon competition favouring the re-establishment and maintenance of the redox homeostasis rather than the synthesis of secondary metabolites at the beginning of ripening, the most ozone responsive developmental stage.

Vitis vinifera encompasses most grapevine cultivars used for table grape and wine production. Unfortunately, this species is highly susceptible to a range of fungal diseases such as downy and powdery mildews and the grey mould, respectively caused by Plasmopara viticola, Erysiphe necator and Botrytis cinerea. Moreover, a complex group of pathogenic fungi that attacks perennial organs is responsible for the so-called grapevine trunk diseases. To overcome the negative impacts of these pathogens on plant development and fruit quality, and avoid excessive crop losses, viticulture needs to perform intense fungicide spraying programs, especially in hot and wet weather conditions. Even organic and biodynamic approaches largely require sulfur- and copper-based formulations that may be detrimental to the soil ecosystem in the long term. Te ecological and environmental sustainability is an increasing concern for consumers and more generally for society. One way to reduce the susceptibility of V. vinifera to pathogens is to breed new cultivars introgressing genetic traits of resistance from American and Asian Vitis spp. Several breeding programs are ongoing in Europe and abroad with an increment of new resistant genotypes available. In parallel to introducing new varieties, which is a long process and ofen not entirely accepted by the market, other strategies like the application of bioactive natural-derived products (silicons, laminarin, potassium phosphonates, analog of salicylic acid, phytomela- 1,2 tonin, etc.) that act as elicitors of plant biotic stress resistance , or the use of ozone (O3) have been proposed as smart approaches to control fungal diseases. Indeed, when applied in aqueous solution, ozone has been shown to suppress spore germination of the esca-associated fungus Phaeoacremonium aleophilum and reduce fungal development by 50% on Cabernet Sauvignon cuttings3. Te use of ozonated water in integrated vineyard pest

1Cátedra de Química Agrícola, E.T.S.I. Agrónomos y de Montes, Universidad de Castilla-La Mancha, Avda. de España s/n, 02071 Albacete, Spain. 2BetterRID (Better Research, Innovation and Development, S.L.), Carretera de Las Peñas (CM‑3203), Km 3.2, Campo de Prácticas‑UCLM, 02071 Albacete, Spain. 3AGAP, CIRAD, INRAe, Institut Agro‑Montpellier SupAgro, Montpellier University, 34060 Montpellier, France. 4Departamento de Ciencia y Tecnología Agroforestal y Genética, Universidad de Castilla-La Mancha, Campus Universitario s/n, 02071 Albacete, Spain. 5These authors contributed equally: Ana Campayo and Stefania Savoi. *email: laurent.torregrosa@ supagro.fr

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Figure 1. Sugar and acid content and organ fresh weight. (a) Malic acid (mEq) as a function of sugar (glucose + fructose, mM). Full coloured circles represent the individual berry or pairs of leaves selected for RNA-Seq; (b) Average concentrations in glucose + fructose (mM), malic acid (mEq), tartaric acid (mEq), and organ fresh weight (g) of the selected triplicates. Error bars represent the SD (n = 3). Selected samples showed no signifcant diferences between conditions (C and OW) according to the independent samples t-test (p < 0.05). Figure was obtained with IBM SPSS Statistics 24 (https://www. ibm. com/ produ cts/ spss- stati stics ).

management appears to be as efective as traditional chemical treatments in reducing fungal populations on leaves and grape bunches 4. Te efciency of ozone is thought to lie in its oxidising potential, which translates into the ability to react with numerous cellular constituents hence a broad-spectrum antimicrobial action5. Its low persistence afer application makes ozone particularly attractive from an environmental point of view. Tis triatomic molecule is highly unstable and spontaneously decomposes into oxygen without leaving hazardous residues, with a shorter half-life in water than in the gaseous state 5. In aqueous solution, ozone can be broken down via a chain reaction mechanism resulting in the production of reactive oxygen species (ROS), · · − · i.e. the hydroperoxide (HO2 ), superoxide ( O2 ) and hydroxyl ( OH) radicals and hydrogen peroxide (H2O2), all contributing to the high oxidising power of ozone6. Ozone enters plant tissues through the stomata, lenticels or physical breaks in the cuticle. Ten it reacts with molecules present in the apoplastic fuid, cell wall and plasma membranes, where it decomposes to produce the ROS mentioned above 7. Under the oxidative stress induced by ozone and derived products, plants develop defence mechanisms at the genetic, transcriptional and biochemical level, which includes the synthesis of antioxidants such as ascorbate, glutathione, enzymes like superoxide dismutases, catalases and peroxidases, and secondary metabolites like carotenoids, terpenoids and phenolics 8–10. When the detoxifcation capacity of plant cells is overwhelmed, cellular damage can occur. Most research about the efects of ozone on plants has focused on the physiological changes triggered by ozone as a pollutant. However, ozone applied in aqueous solution and in a timely manner is expected to interact with plants diferently than in the gaseous state, with a sufciently high phytotoxic threshold that allows its incorpo- ration in irrigation and spraying treatments in diferent crop species11. Unfortunately, literature concerning the efects of ozonated water on grapevine plants is scarce and almost exclusively dedicated to analysing its efect on microbial populations3,4,12, except a few recent studies describing its impact on grape and wine composition12–16. Te microvine is a convenient model plant for performing physiological studies in a semi-controlled environment. Carrying the Vvigai1 mutation, microvines exhibit a continuous fowering, simultaneously displaying all the successive stages of fruit development on a single shoot17. Tis model has already facilitated transcriptomics approaches of the circadian cycle 18, high-temperature stresses 19,20, metabolomics works surveying glycosylated aroma precursors21,22, and several berry developmental studies23–25. In this study, this model allowed us to characterise the early transcriptome changes triggered in grapevine leaves and berries at diferent ripening stages afer in planta sprayings of ozonated water solutions. Results The balance in primary metabolites: an analytical tool to select RNA‑Seq samples. At the beginning of ripening (BR), sof green berries were sampled while still in the lag phase with no visible anthocyanin accumulation in their skin. Tese berries just started to accumulate sugar while consuming malate (Fig. 1a). As expected, berries in the mid-ripening stage (MR) showed higher sugar concentrations (close to 1 M) and a lower amount of malic acid (Fig. 1a). Mature leaf samples (L) displayed a comparable amount of soluble sugars

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Figure 2. Transcriptomics overview. Principal component analysis of the transcriptomic samples in (a) leaves (L, green), (b) berries at the beginning of ripening (BR, orange), and (c) berries in mid-ripening (MR, purple); (d) sample dendrogram, with C and OW samples represented with a square and a triangle, respectively; (e) number of DEGs in L, BR and MR, and (f) commonly and uniquely modulated genes.

to BR, with a two-fold lower malate concentration, indicating strong diferentiation between the source (leaves) and sink (berries) organs. Tanks to the measurements of sugars and acids, it was possible to gather synchronised samples26 for further RNA-Seq analysis with the aim to reduce biases in gene expression caused by the natural developmental asynchrony of grapevine berries and focus only on the early transcriptomic changes triggered by the ozonated water treatment. Indeed, biological triplicates were selected at the same sugar (glucose + fructose) concentrations for control (C) and ozonated water treatment (OW), namely 158 mM in L, 291 mM in BR, and 864 mM in MR (Fig. 1b). Malic and tartaric acids were 184 mEq and 255 mEq for L, 363 mEq and 120 mEq for BR, and 139 mEq and 103 mEq for MR (Fig. 1b), giving an average malate/tartrate ratio of 0.7, 3.0, and 1.3, respectively in L, BR and MR. No signifcant diferences were found between conditions for sugar, acids and sample weight (Fig. 1b).

Transcriptomic overview in leaf and ripening berry. Principal component analyses were performed to visualise the global transcriptome trends (Fig. 2a–c). Te frst two principal components (PC1 + PC2) explained 65, 74, and 63% of the variance among samples in L, BR, and MR. C and OW samples were clearly resolved in BR, while the separation was less obvious in L and MR. Te hierarchical clustering dendrogram showed the degree of similarity between the transcriptome profle of all the samples analysed (Fig. 2d). Te most striking diferences in the transcriptome were determined by the type of organ, i.e. leaf or berry, followed by the berry developmental stage. As before, the dendrogram showed that the C and OW BR samples grouped separately, while for L and MR the three OW replicates clustered conjointly, but one C sample was placed in a diferent branch than the other two. Genes diferentially expressed according to the ozone treatment were tested in the leaves and two berry developmental stages separately (Fig. 2e,f, and Supplementary Table S1). In L, the total number of DEGs was 191, with 84 up-regulated genes and 107 down-regulated. Te most intense response to the treatment was observed in BR with 2006 DEGs, of which 1021 were up-regulated and 985 down-regulated. In MR, the treatment modulated the expression of 275 genes, with 207 up-regulated and 68 down-regulated. Tere were 43 commonly regulated DEGs between all the organs analysed, of which 40 up-regulated and 3 down-regulated. Te DEGs commonly up-regulated in L, BR, and MR were mostly genes encoding heat shock proteins and chaperones, in addition to heat-stress transcription factor and galactinol synthase. Other genes among the up-regulated ones were a malate

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Figure 3. Signifcantly enriched GO categories in the up-regulated genes in (a) leaves, (b) berries at the beginning of ripening and (c) berries in mid-ripening, divided in biological process (BP, green), cellular component (CC, orange), and molecular function (MF, purple) expressed as − log10(adjusted p value).

dehydrogenase, an argonaute protein, a RuBisCO large subunit-binding protein, several peptidyl-prolyl cis–trans isomerases, a calcyclin-binding protein, a NAD transporter and a putative SERF-like protein. Conversely, in the commonly down-regulated DEGs, we observed an auxin transporter-like protein and a pectin methylesterase (Supplementary Table S1). Te lists of up- and down-regulated genes were separately screened for signifcant enrichment (p < 0.001) in Gene Ontology (GO) categories in the Biological Process (BP), Cellular Component (CC), and Molecular Func- tion (MF). Te down-regulated genes fell in a limited number of enriched categories: for example, in L there were two CC categories such as cell wall and external encapsulating structure, in BR only regulation of gene expression for the BP, while no categories were enriched in MR. Instead, a higher number of categories and subcategories were detected in the up-regulated genes (Fig. 3). In common to the three organs, several categories reported enrichment for protein folding and related categories, and response to a plethora of stresses including response to heat, response to hydrogen peroxide, response to reactive oxygen species, and response to oxidative stress (Fig. 3).

Up‑regulation of heat‑shock proteins and chaperones: a common response of leaves and ber‑ ries. To prioritise the genes whose expression changed the most afer a 90-min exposure to ozone, each list of DEGs was ranked according to their respective absolute changes in expression between C and OW (Supple- mentary Table S1). Te overall reaction in both leaves and berries was primarily to activate rather than repress physiological processes. Te highest number of genes up-regulated by the OW treatment belonged to heat shock proteins (HSPs) and chaperones, with 11 small HSP or HSP20 genes upregulated in L, 36 in BR and 28 in MR (Fig. 4). Among the HSP20 recently identifed in grapevine27, VviHSP20-09, VviHSP20-17, VviHSP20-22, VviHSP20-25, VviHSP20-27, VviHSP20-35, VviHSP20-36, VviHSP20-39, VviHSP20-42, and VviHSP20-44 were commonly highly up-regulated. Other HSPs of higher molecular weight such as HSP70 and HSP90 were up-regulated as well, together with a series of chaperones, such as the DNAJ homolog, calnexin and calreticulin (Fig. 4). Interestingly, diverse heat-stress transcription factors (HSFs) were also modulated by the treatment: VviHSF- A6b was up-regulated in L, BR, and MR. VviHSF-A2 and VviHSF-B2a were up-regulated only in the berry (BR and MR). VviHSF-A3 was up-regulated in BR, VviHSF-B2b in MR, and VviHSF-A1b was the only gene down-regulated in BR. Moreover, the transcription factors multiprotein-bridging factor 1c and 1a (VviMBF1C, VviMBF1A) were up-regulated, with a noticeably strong activation of the frst one in the berries. Lastly, as part of the stress response, two galactinol synthases (VviGOLS) were up-regulated in L, BR, and MR (Fig. 4).

Antioxidant homeostasis. Other categories of DEGs, identifed mostly in BR, were related to the antioxidant homeostasis, which involves the scavenging of the reactive oxygen species (ROS). Te ascorbate–glutathione cycle (AsA-GSH cycle) detoxifes ROS through the activity of ascorbate peroxidase (APX), monode- hydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), requiring a pool of ascorbate, glutathione and NADPH. Here, one APX (Vitvi08g01143) in BR and one DHAR (Vitvi13g00241) in L and BR were up-regulated (Fig. 5), indicating an enhanced turnover of the cycle under stress to scavenge O3-generated H2O2 into water. Paradoxically, two isogenes encoding VTC2 (GDP-L-galactose phosphorylase), a regulatory step in AsA biosynthesis, were down-regulated in BR (Fig. 5). Other antioxidant enzymes such as glutathione S-transferases (GSTs), catalases (CATs), peroxidases (PODs), superoxide dismutases (SODs) and redoxins (RXs) were modulated by the stress (Fig. 5). In particular, 9 GSTs were up-regulated (6 in BR and 5 in MR), while three were down-regulated in BR. Genes generally annotated

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Figure 4. Heat-shock proteins, chaperones and related transcription factors modulated by the ozonated water treatment in leaves (lef column), berries at the beginning of ripening (middle column) and berries in mid- ripening (right column) are represented in heatmaps as log2FC(OW/C). Red and blue colours indicate up- or down-regulation, respectively. Te coloured sidebar on the lef displays the class of genes.

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Figure 5. Oxidative stress-related genes modulated by the ozonated water treatment in leaves (lef column), berries at the beginning of ripening (middle column) and berries in mid-ripening (right column) are represented in heatmaps as log2FC(OW/C). Red and blue colours indicate up- or down-regulation, respectively. Te coloured sidebar on the lef displays the class of genes.

as CAT (3), POD (4), chaperone for SOD (1), and RX (19) were modulated by OW in the BR berry with some genes induced and others repressed.

Intense down‑regulation of the cell wall‑related genes and plasma membrane aquapor‑ ins. Ozonated water sprayed all over the plant surface strongly impacted the cuticle and cell wall-related genes of leaves and berries with more emphasis on the BR berry (Fig. 6). Six cuticle genes were down-regulated together with ten expansins, among which the most highly repressed were VviEXPA11, VviEXPA14, VviEXPA18, and VviEXPA19. Four cellulose synthases and ten xyloglucan endotransglucosylase / hydrolase genes were up- regulated, while seven cellulose synthase-like were down-regulated together with three pectate lyases and other pectinesterases. Interestingly, four aquaporins were modulated by OW at the beginning of ripening: VviPIP1.1 and VviPIP2.3 located on the plasma membrane were down-regulated; on the contrary VviTIP2.1 and VviTIP3.1, whose locali- sation is the tonoplast, were up-regulated.

Secondary metabolism is afected only at the beginning of ripening. Antioxidant secondary metabolites like carotenoids, terpenoids and phenolic compounds can be synthesised in response to the stress. Te expression level of several related genes was modulated by OW in BR berries, while no signifcant impact could be detected in L and MR (Fig. 7). Genes involved in the early steps of carotenoid synthesis in grapevines such as a 15-cis-ζ-carotene isomerase (VviZISO1), a ζ-carotene desaturase (VviZDS1), a carotenoid isomerase (VviCISO1), and a lycopene β-cyclase (VviLBCY2) were down-regulated by OW in BR. Interestingly, a violaxanthin de-epoxidase (VviVDE2), involved in the violaxanthin and lutein epoxide (xanthophyll) cycles, was up-regulated. Carotenoids can be cleaved through carotenoid cleavage dioxygenases (CDD) to form volatile favour and aroma compounds such as the C13-norisoprenoids (e.g. β-ionone and β-damascenone). Te isoform VviCCD4b was up-regulated in BR, while VviCCD4a was down-regulated in MR. Lastly, neoxanthin and violaxanthin can also be cleaved by 9-cis-epoxyca- rotenoid dioxygenase (NCED) to form the hormone ABA; the three VviNCED were modulated with VviNCED1 up-regulated, while VviNCED2 and VviNCED3 were down-regulated together with a xanthoxin dehydrogenase (VviABA2) in BR.

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Figure 6. Cell wall and growth-related genes modulated by the ozonated water treatment in leaves (lef column), berries at the beginning of ripening (middle column) and berries in mid-ripening (right column) are represented in heatmaps as log2FC(OW/C). Red and blue colours indicate up- or down-regulation, respectively. Te coloured sidebar on the lef displays the class of genes. XTH: xyloglucan endotransglucosylase/hydrolase.

Plant terpenoids are synthesised via the cytosolic MVA and the plastidial MEP pathways. In the MVA, one acetyl-CoA acetyltransferase (VviAACT) was down-regulated in ozonated BR, while a hydroxymethylglutaryl- CoA reductase (VviHMGR) was up-regulated. In the MEP, the genes encoding a 1-deoxy-D-xylulose-5-phosphate synthase (VviDXS), a geranylgeranyl pyrophosphate synthase (VviGGPPS), and a terpene synthase (VviTPS31) were down-regulated in BR. Several genes of the phenylpropanoid and favonoid pathway were also diferentially expressed by OW. In particular, two phenylalanine ammonia-lyases (VviPAL) and one trans-cinnamate-4-monooxygenase (VviC4H) were down-regulated in BR. In contrast, three cafeic acid 3-O-methyltransferases (VviCOMT) were diferentially regulated with two genes down-regulated and one up-regulated. Other genes involved in the terminal steps of monolignol biosynthesis were also afected; namely, a cinnamoyl-CoA reductase (VviCCR) was down-regulated, while a cinnamyl alcohol dehydrogenase (VviCAD) up-regulated. In the same berries, two isofavone reductases (VviIFR), implicated in the isofavonoid phytoalexin branch pathway, were down-regulated. Lastly, the ozonation of MR berries only reduced the expression of a hydroxycinnamoyl-CoA: shikimate / quinate hydroxycinnamoyl- transferase (VviHCT) and a VviIFR. Regarding the favonoid pathway, a favanone 3-hydroxylase (VviF3H), a favonoid 3′-hydroxylase (VviF3′Ha) and a leucoanthocyanidin dioxygenase (VviLDOX) were modulated by the stress in BR: the frst one up-regulated, while the last two down-regulated. A cafeoyl shikimate esterase (VviCSE) and the VviF3′Ha were the only DEGs down-regulated in L. Lastly, relevant transcription factors, such as VviMYB5a, VviMYB5b, and VviMYBF1, controlling diferent branches of the favonoid pathway, were down-regulated in BR.

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Figure 7. Secondary metabolites genes modulated by the ozonated water treatment in leaves (lef column), berries at the beginning of ripening (middle column) and berries in mid-ripening (right column) are represented in heatmaps as log2FC(OW/C). Red and blue colours indicate up- or down-regulation, respectively. Te coloured sidebar on the lef displays the class of genes.

Discussion Although the major use of ozone in agriculture lies in its antifungal activities, as confrmed in grapevine3,412, there is still a lack of information on how ozone can afect grapevine physiology and grape composition. Besides, the role of ozone in preventing or controlling infections of Plasmopara viticola and Erysiphe necator, the agents responsible for downy and powdery mildews in grapevine, has not yet been confrmed. Previous feld experiments reported versatile impacts of ozonated water sprayings on the composition in phenolic and terpenoid compounds of grapes and resulting wines 12–14,16. Such discrepancies suggest that more studies in controlled conditions (e.g. with potted plants grown in greenhouses) are needed to describe the molecular and biochemical changes induced by O 3 in grapevine organs. Using the microvine model, this study represents the frst transcriptomics analysis exploring the responses triggered by ozonated water spraying on grapevine leaves and fruits. BR berries appeared incredibly responsive to ozone exhibiting the highest number of DEGs. Te intense transcriptomic reprogramming at the onset of ripening, largely documented in grapevine fruit 28,29, has also been associated with ROS accumulation30, whose synthesis occurs most intensively during the night 18. Due to the method of monitoring the development of the berries and their sampling, we can reasonably assume that BR berries were very close to the H 2O2 and catalase peaks that were spotted in non-developmentally synchronised fruits30. Te intense transcriptomic changes described here showed that endogenous ROS production previously reported at the onset of ripening is actually far from saturating in standard conditions with no stress. Te observed response can also be explained by the greater variety of reactive species formed from aqueous O3, including the more potent oxidant and chain-propagating hydroxyl radical 6, which can difer from the ones endogenously produced. In fact, the endogenous ripening related ROS production does not result in the cell wall and growth inhibition, as this production is suspected to occur just before or at the inception of the second fruit growth phase. Indeed, recent physiological and transcriptomic works evidenced that the less harmful hydrogen 31,32 peroxide (H 2O2) accelerated ripening in Kyoho variety . Te genes suggested by the authors to induce the early ripening were associated with the oxidative stress, photosynthesis, cell wall deacetylation and degradation. More studies are needed to decipher the possible role of ozonated water in grape ripening, knowing that H2O2 is only one of the ROS formed by the decomposition of ozone in aqueous solution6. Berry sofening marks the onset of the massive import of sugars in grapevine. Surprisingly, VviSWEET10, which is implicated in the unloading of phloem sucrose inside the berry 25, was up-regulated in BR (Supplementary Table S1) together with two TIPs, aquaporins of the vacuole. But the expression of VviHT6, the major sucrose transporter on the tonoplast, was not afected, leaving open the question of a possible enhancement of the ripening program under ozonated water. As ozone decomposition strongly depends on pH, its decay may be faster in the cell wall and cytoplasm than in the acidic vacuole of berries at the beginning of ripening 33.

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In our dataset based on developmentally synchronised berries, some cuticle related genes were down-regulated. Te degradation of this protective barrier, which leads to greater penetration of ozone into the plant cells, has been reported in growing plants and postharvest fruits exposed to ozone 34. Moreover, key expansins involved in the cellular expansion and growth 35 were down-regulated with pectate lyases, pectinesterases and cellulose synthase-like genes indicating an immediate multifaceted efect unsettling the cell-wall dynamics, further exacerbated by the down-regulation of two plasma membrane aquaporins suggesting a limited water infux. Although all point toward a decrease in cell expansion, we did not observe any specifc phenotypes on leaf or grape development (data not shown) in the following weeks afer the treatment. Tis might indicate that ozone triggers only a transitory inhibition of cell wall remodelling and expansion. However, ozone has been shown to modify the composition and mechanical properties of grape skin cell walls 36, afecting aroma and polyphenols extraction during winemaking 37. Te lower anthocyanin extractability observed afer spraying ozonated water on grapevines13,15 may originate from the down-regulation of genes encoding pectin-degrading enzymes detected in ozonated berries. Te frst coordinated response to the ozonated treatment was the induction of a plethora of HSPs and other chaperones. HSPs are involved in the cellular response to a diverse array of stresses, including oxidative 38. Tey act mainly as molecular chaperones, participating in protein folding, assembly, translocation and degradation in many normal cellular processes and maintain proteins in their functional conformations under stress conditions, preventing their aggregation and denaturation, and assisting in protein refolding39. Te induction of HSP transcripts in plants fumigated with ozone was frst described in parsley 40 and then confrmed in other plants such as Arabidopsis thaliana and Medicago truncatula41,42. Using proteomic approaches, the increased expression of these proteins under ozone stress was also detected in poplar, bean, maize and rice 43–45. Te induction of HSPs is under the tight control of an HSF network46, with signifcant players VviHSF-A2 and VviHSF-A6b already reported intensifed in grapevine under stress19, 20,47, ofen together with VviGOLS48. Moreover, transgenic Arabidopsis thaliana plants constitutively expressing the transcriptional coactivator AtMBF1c showed enhanced tolerance to environmental stresses 49. Here these genes were strongly up-regulated, possibly cross-regulating several plant response mechanisms to various stresses. Plants submitted to abiotic or biotic stresses typically produce ROS, triggering oxidative stress 50,51. AsA is the most abundant antioxidant in plant cells, found in all subcellular compartments, including the apoplast, and therefore representing the frst line of defence against ozone 52. AsA can directly scavenge ozone and diferent 53 ROS and, along with glutathione in the AsA-GSH cycle, is the primary H2O2 reducing substrate operating in cytosol, chloroplasts and mitochondria of plant cells54. It has been shown that the antioxidant response to the stress is genotype-dependent, with grape varieties such as Touriga Nacional able to boost the cell redox‐bufering capacity with the existing AsA and GSH pools, while other varieties, like Trincadeira, need to synthesise both metabolites because of its incapacity to keep the cellular redox state at working levels 55. Terefore, it is not surprising that VviVTC2, the central regulator of the AsA biosynthetic pathway56, was down-regulated in BR, indicating a non-need for resynthesis but a bufering capacity of the microvine coping with oxidative stress. Similarly to our results, OsVTC2 was down-regulated in ozone-exposed rice, attributing the changes in total and reduced AsA concentration to AsA turnover rather than biosynthesis, with a parallel increase of OsAPX, OsDHAR, and OsGR57. Also in our dataset, VviAPX and VviDHAR were up-regulated under ozone. Elevated expression of these two genes in response to ozone has already been detected in Arabidopsis thaliana58,59, and DHAR-overexpressing plants have shown increased tolerance to ozone by incrementing foliar AsA level60. In grapevine, AsA is also a precursor for the synthesis of both tartaric and oxalic acids. Te down-regulation of VviVTC2 in BR berry under ozone stress could indicate a switch from the Smirnof-Wheeler (SW) pathway to the alternative AsA biosynthetic pathway, knowing that the frst one supports AsA biosynthesis in immature berries, while the alternative synthesises AsA from a methyl derivative of D-galacturonic acid released during pectin degradation as fruits ripen61. Given that GDP-D-mannose and GDP-L-galactose, intermediates of the SW pathway, are also precursors of the non-cellulosic components of the plant cell wall62, we can speculate that the inhibition of enzymes involved in cell wall synthesis and growth would lead to AsA sparing and in turn to reduced AsA synthesis, materialised through the down-regulation of VviVTC2. Other critical antioxidant enzymes such as CAT, POD, SOD, RX, and GST were modulated by the stress indicating an intense redox homeostasis activity to prevent damage from ozone and its by-products50. In particular, the treatment induced the expression of six out of nine GSTs detected in BR berries. Tis elicitor efect was also observed in MR berries, confrming previous results in ozone-exposed Arabidopsis and rice seedlings 41,45,63. Tiols such as GSH are versatile targets for most oxidants, including ozone64, so we hypothesise that GST transcript levels increased in order to counterbalance reduced substrate availability. In addition, this alleged reduced availability of GSH may have been enhanced in the BR berry by VviDHAR up-regulation. GSTs are also necessary for the transport of anthocyanins from the cytosol to the vacuole. Consequently, a strong correlation between these proteins and anthocyanin accumulation has been found in V. vinifera65, indicating a possible involvement in the increased phenolic content under ozonated water treatments. Although many secondary metabolites are important antioxidants whose synthesis is typically induced in plants as a defence mechanism against ozone 8, 9, in the early transcriptional response to the ozonated water application their pathways were generally unafected in leaves and mid-ripening berries, with some genes down- regulated in berries starting to ripen. Carotenoids contribute to light harvesting and protect the photosynthetic membrane against photo-oxidative damage, not only by quenching the triplet states of chlorophyll but also by scavenging ROS 66. A possible impair- ment of carotenoid synthesis through the down-regulation of VviZISO1, VviZDS1, VviCISO1 and VviLBCY2 in the early ripening berry seems counter-intuitive, however, similar observations were made in diferent rice genotypes57. Te regeneration of carotenes and xanthophylls from their oxidised radicals relies on AsA53 and, in addition, the violaxanthin de-epoxidase enzyme requires AsA as a cofactor 67. Here, the higher expression of

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VviVDE2 in the ozonated BR berry indicates an activation of the de-epoxidation in the xanthophyll cycles, which protects against ROS-generating stresses68. Tis mechanism is expected to be also activated in ozone-treated leaves as they ofen undergo a reduction in photosynthetic rates and need to dissipate the excess excitation energy absorbed by the antennae10. However, here, no sign of photosynthetic apparatus damage was observed in leaves. Te activation of the xanthophyll cycles in BR berry may respond to the zeaxanthin and lutein roles in ROS scavenging and preventing membrane lipid peroxidation 69,70. Terpenoids have been shown to improve the ability of plants to cope with internal oxidative changes71, reduce ozone damage and quench ozone and ROS 9. However, ozone has been shown to stimulate and reduce the biosynthesis and emission of these volatiles depending on the severity and duration of the exposure and the plant species sensitivity 72. Here, the overall down-regulation of the genes involved in their synthesis in the treated BR berries, such as VviDXS and VviTPS31, key determinants in the production of monoterpenes in grapevines73,74, and VviGGPPS, the precursor of diterpenes and carotenoids, contrasts with the higher terpenoid content found in berries from Bobal and Vermentino grapevines subjected to ozonated water treatments12, 13. Nevertheless, this increase was detected in berries at the end of the ripening period and not afer each ozone exposure, and was much less pronounced when the treatment was applied at the onset of veraison 13. It should be noted however, that the transcriptional profles reported in the current study represent an early response, while longer-term efects on expression (e.g. at the time of harvest) could vary. In this line, an immediate depression of isoprene emission was reported in Quercus pubescens leaves exposed to ozone, attributed to a temporary inhibition of photosynthesis, but a subsequent fast recovery and even stimulation 12 days afer fumigation 75. Plants exposed to ozone ofen respond with increased transcription and activities of enzymes involved in the phenylpropanoid, lignin and favonoid pathways because of their barrier and antioxidant roles10,76. However, this response may not be immediate: for example, the induction of genes involved in the favonoid synthesis in Arabidopsis was part of the later response to two days of ozone exposure, with chalcone synthase, dihydrofavonol reductase and leucoanthocyanidin dioxygenase being the most responsive 41. In Melissa ofcinalis L., an ozone treatment (5 h) initially impaired PAL activity, the frst enzyme in the general phenylpropanoid pathway, followed by a subsequent increase 7 h afer the end of the exposure 77. Similarly, our results showed that the early response to the ozonated water treatment, mainly in BR berries, consisted of an overall down-regulation of several genes involved in these pathways. Whether these genes are reactivated later is presently unknown. Plants are sessile organisms that produce metabolites as an adaptive strategy to cope with challenging and changing environments78. Secondary metabolic routes are highly demanding for energy and carbon compounds, including the metabolites synthesis, their transcriptional regulation, and transport in subcellular compartments 79. On the urgency to respond to the stress in the short-time, grapevine vegetative and reproductive organs appar- ently prefer to allocate carbon and energy to immediate defence response (HSPs, chaperones, AsA-GSH cycle). We can speculate that multiple treatments and/or a longer span between ozone exposure and sampling could lead to adaptation mechanisms triggering cascades of signal networks ending with the synthesis of stress-related genes and secondary metabolites accumulation, as ofen observed in grapes at harvest. Tis study is an original contribution performed with a perennial fruit crop. Te goal was to characterise the frst responses of both vegetative organs and feshy fruits to ozonated water treatments (Supplementary Figure S1). Terefore, further studies will be needed to get a comprehensive understanding of the long-term efects on plant physiology and especially on fruit composition. Based on this frst study and previous experiences 17, we propose the microvine as a relevant perennial feshy fruit model to perform such investigations. Methods Plant material. Two-year-old ML1 microvines were grown in 3 L pots under semi-controlled conditions in a greenhouse (Montpellier SupAgro- INRAe campus, France) with day/night temperature 25/15 °C, 1 kPa of VPD, and 12 h photoperiod. Microvine ML1, which was obtained by L. Torregrosa17, complies with relevant institutional, national, and international guidelines and legislation. Plants were managed for eight months to display all fruit developmental stages from fowering to ripe stages 17. Plants were maintained at full ETP (Evapo- Transpiration Potential), thus avoiding water stress issues, and no fungicide sprayings were performed. At the beginning of the experiment, the developmental stage of single green berries was checked by visual inspection and frmness assessment23, in order to detect the frst sofening signs as the onset of sugar storage. Single berry growth was weekly monitored by image analysis of clusters taken 30 internodes below the apex, with a Lumix FZ100 camera (Panasonic).

Ozonated water treatment and sampling. Before treatment, plants were randomly divided into two groups: four plants for the control (C) and four plants for the ozonated water treatment (OW) (Fig. 8a). To have ventilation representative of feld conditions and prevent of-target ozone difusion, plants were brought outside the greenhouse for the entire duration of the experiment (9 am–12 pm), all C and OW plants being managed in the same environmental conditions. To avoid additional stress (e.g. excessive temperature or light exposure), the experiment was done in the morning at a time when the temperature was similar to that of the greenhouse (25 °C) and the plants were kept in the shade. In addition, the plants were maintained outside for about one hour prior to treatment to allow them to adapt to the outdoor environment. Ozonated water was prepared extempo- raneously using an ozone generator (Cosemar Ozono S.L., Spain) connected to a sprayer containing Milli-Q water at a temperature of 15 °C and a conductivity of 18.2 MΩ/cm. A redox meter (PCE-228-R, PCE Ibérica S.L., Spain) was used to continuously measure in millivolts (mV) the oxidation–reduction potential (ORP) of the aqueous solution. One hundred ffy mL of ozonated water (once its ORP reached 1000 mV) was sprayed on the entire surface of each OW plant (Fig. 8b). Te four C vines were sprayed with the same amount of Milli-Q water used for the treatment. Immediately afer the spraying, plants were enclosed in plastic bags to prevent drif and

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Figure 8. Experimental setup. (a) Microvine plants in the greenhouse; (b) ozonated water spraying treatment on plants showing diferent berry developmental stages; (c) microvine covered with a plastic bag immediately afer the treatment.

avoid too rapid ozonated water evaporation (Fig. 8c), in an attempt to mimic feld conditions where the spraying is usually carried out early in the morning, at low wind. Ninety minutes afer the start of the treatment, 15 single individual green berries at the beginning of ripening (BR) (+ 3 days afer sofening), 15 single individual berries in the mid-ripening stage (MR) (+ 18 days afer sofening), and two adult leaves per plant (L) located between the 30th and 40th nodes were sampled for both C and OW. Single berry samples (pericarp and seeds) and leaves were wrapped separately in aluminium foils and immediately frozen in liquid N 2. Each sample was weighed and ground into liquid N 2 using a ball mill (Retsch, Germany). Te resulting powder was stored at − 80 °C, and used for primary metabolites and RNA analyses.

Primary metabolites analysis. Sugars and acids were analysed by high-performance liquid chromatog- raphy (HPLC), according to Rienth et al.20. Briefy, 100 mg of leaf or berry frozen powder was 5 × diluted in HCl 0.25 N and lef overnight at room temperature afer shaking. Samples were then centrifuged at 15,000 g for 10 min, and a supernatant aliquot was diluted 10 × with a solution of H 2SO4 5 mM containing 600 µM acetic acid as internal standard, before injection into the HPLC system. Te statistical analysis of the data was performed with SPSS statistics sofware (version 23.0 for Windows, Chicago, IL, USA). Te mean values of the selected samples were compared using the independent samples t-test, and the diferences were considered statistically signifcant when the p-value < 0.05.

RNA extraction and sequencing. Tree samples per treatment (C and OW) and organ (L, BR and MR) were selected for individual RNA extraction and library preparation as described in Rienth et al.80. Samples were sequenced on an Illumina HiSeq3000 in paired-end mode, 2 × 150 bp reads, at the Genotoul platform of INRAe- Toulouse (France).

Data analysis. Raw reads were trimmed for quality and length with Trimmomatic, version 0.3881. Reads were aligned against the reference grapevine genome PN40024 12X282, using the sofware Hisat2, version 2.1.083 with standard parameters, yielding an average of 25.3 M sequence per sample (Supplementary Table S2). Aligned reads were counted using the VCost.v3 annotation with HTSeq-count (version 0.9.1)84, in union mode, mRNA type, nonunique all, and stranded options. Only genes with RPKM > 1 were kept for further analysis (Supplemen- tary Table S3). Diferentially expressed genes (DEGs) (FDR < 0.05) were screened with the R package DeSeq2 85. Overrepresented gene categories were identifed with the gProfler web-server (version 101_eg48_p14_baf17f0) with a signifcance threshold of 0.001. PCA and dendogram fgures were drawn with RStudio (package ggplot2). Heat map fgures were drawn with RStudio (package pheatmap v1.0.12). Data availability Raw transcriptomics reads have been deposited in NCBI Sequence Read Archive (http://www. ncbi. nlm. nih. gov/ sra). Te BioProject is PRJNA678610.

Received: 23 January 2021; Accepted: 25 March 2021

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References 1. Pagliarani, C. et al. Te molecular priming of defense responses is diferently regulated in grapevine genotypes following elicitor application against powdery mildew. Int. J. Mol. Sci. 21, 6776 (2020). 2. Zhao, D., Wang, H., Chen, S., Yu, D. & Reiter, R. J. Phytomelatonin: an emerging regulator of plant biotic stress resistance. Trends Plant Sci. 26, 70–82 (2021). 3. Pierron, R. J. G. et al. In vitro and in planta fungicide properties of ozonated water against the esca-associated fungus Phaeoacr- emonium aleophilum. Sci. Hortic. 189, 184–191 (2015). 4. Raio, A., Feliciani, A., Ferri, V. & Carboni, C. Integrated vineyard management trials using ozonated and electrolized water. Infowine Internet J. Enol. Vitic. 2(6), 1–6 (2016). 5. Khadre, M. A., Yousef, A. E. & Kim, J.-G. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242–1252 (2001). 6. Hoigné, J. Chemistry of aqueous ozone and transformation of pollutants by ozonation and advanced oxidation processes. In Quality and Treatment of Drinking Water (ed. Hrubec, J.) 83–141 (Springer, 1998). https://doi.org/10.1007/978-3-540-68089-5_5. 7. Forney, C. F. Postharvest response of horticultural products to ozone. In Postharvest Oxidative Stress in Horticultural Crops (ed. Hodges, D. M.) 13–43 (Food Products Press, 2003). 8. Heath, R. L. Modifcation of the biochemical pathways of plants induced by ozone: What are the varied routes to change?. Environ. Pollut. 155, 453–463 (2008). 9. Loreto, F. & Schnitzler, J. P. Abiotic stresses and induced BVOCs. Trends Plant Sci. 15, 154–166 (2010). 10. Castagna, A. & Ranieri, A. Detoxifcation and repair process of ozone injury: from O3 uptake to gene expression adjustment. Environ. Pollut. 157, 1461–1469 (2009). 11. Graham, T., Zhang, P., Zheng, Y. & Dixon, M. A. Phytotoxicity of aqueous ozone on fve grown nursery species. HortScience 44, 774–780 (2009). 12. Modesti, M. et al. Efects of treatments with ozonated water in the vineyard (cv Vermentino) on microbial population and fruit quality parameters. BIO Web Conf. 13, 04011 (2019). 13. Campayo, A. et al. Spraying ozonated water on Bobal grapevines: efect on grape quality. Food Res. Int. 125, 108540 (2019). 14. Campayo, A., Serrano de la Hoz, K., García-Martínez, M. M., Salinas, M. R. & Alonso, G. L. Spraying ozonated water on Bobal grapevines: efect on wine quality. Biomolecules 10, 213 (2020). 15. Campayo, A., Serrano de la Hoz, K., García-Martínez, M. M., Salinas, M. R. & Alonso, G. L. Novel endotherapy-based applications of ozonated water to Bobal grapevines: efect on grape quality. Agronomy 10, 1218 (2020). 16. García-Martínez, M. M. et al. Oenological characteristics of Vitis vinifera L. Cabernet Sauvignon grapes from vineyards treated with ozonated water. Aust. J. Grape Wine Res. https://doi.org/10.1111/ajgw.12454 (2020). 17. Torregrosa, L., Rienth, M., Romieu, C. & Pellegrino, A. Te microvine, a model for studies in grapevine physiology and genetics. OENO One 53, 373–391 (2019). 18. Rienth, M. et al. Is transcriptomic regulation of berry development more important at night than during the day?. PLoS ONE 9, e88844 (2014). 19. Rienth, M. et al. Day and night heat stress trigger diferent transcriptomic responses in green and ripening grapevine (Vitis vinifera) fruit. BMC Plant Biol. 14, 108 (2014). 20. Rienth, M. et al. Temperature desynchronizes sugar and organic acid metabolism in ripening grapevine fruits and remodels their transcriptome. BMC Plant Biol. 16, 164 (2016). 21. Sánchez-Gómez, R. et al. Te Microvine, a plant model to study the efect of vine-shoot extract on the accumulation of glycosylated aroma precursors in grapes. J. Sci. Food Agric. 98, 3031–3040 (2018). 22. Sánchez-Gómez, R. et al. Behavior of glycosylated aroma precursors in Microvine fruits afer guaiacol foliar application. Sci. Hortic. 246, e1–e8 (2019). 23. Bigard, A. et al. Vitis vinifera L. fruit diversity to breed varieties anticipating climate changes. Front. Plant Sci. 9, 455 (2018). 24. Bigard, A., Romieu, C., Sire, Y. & Torregrosa, L. Vitis vinifera L. diversity for cations and acidity is suitable for breeding fruits coping with climate warming. Front. Plant Sci. 11, 01175 (2020). 25. Savoi, S., Torregrosa, L. & Romieu, C. Transcripts repressed at the stop of phloem unloading highlight the energy efciency of sugar import in the ripening V. vinifera fruit. bioRxiv 2021.01.19.427234, https://doi.org/10.1101/2021.01.19.427234 (2021). 26. Shahood, R., Torregrosa, L., Savoi, S. & Romieu, C. First quantitative assessment of growth, sugar accumulation and malate breakdown in a single ripening berry. OENO One 54, 1077–1092 (2020). 27. Ji, X.-R., Yu, Y.-H., Ni, P.-Y., Zhang, G.-H. & Guo, D.-L. Genome-wide identifcation of small heat-shock protein (HSP20) gene family in grape and expression profle during berry development. BMC Plant Biol. 19, 433 (2019). 28. Fasoli, M. et al. Te grapevine expression atlas reveals a deep transcriptome shif driving the entire plant into a maturation program. Plant Cell 24, 3489–3505 (2012). 29. Fasoli, M. et al. Timing and order of the molecular events marking the onset of berry ripening in grapevine. Plant Physiol. 178, 1187–1206 (2018). 30. Pilati, S. et al. Te onset of grapevine berry ripening is characterized by ROS accumulation and lipoxygenase-mediated membrane peroxidation in the skin. BMC Plant Biol. 14, 87 (2014). 31. Guo, D. L. et al. Hydrogen peroxide treatment promotes early ripening of Kyoho grape. Aust. J. Grape Wine Res. 25, 357–362 (2019). 32. Guo, D. L., Wang, Z., Pei, M.-S., Guo, L.-L. & Yu, Y.-H. Transcriptome analysis reveals mechanism of early ripening in Kyoho grape with hydrogen peroxide treatment. BMC Genomics 21, 784 (2020). 33. Gardoni, D., Vailati, A. & Canziani, R. Decay of ozone in water: A review. Ozone Sci. Eng. 34, 233–242 (2012). 34. Hodges, D. M. Postharvest oxidative stress in horticultural crops (CRC Press, 2003). 35. Dal Santo, S. et al. Genome-wide analysis of the expansin gene superfamily reveals grapevine-specifc structural and functional characteristics. PLoS ONE 8, e62206 (2013). 36. Paissoni, M. A. et al. Impact of post-harvest ozone treatments on the skin phenolic extractability of red winegrapes cv Barbera and Nebbiolo (Vitis vinifera L.). Food Res. Int. 98, 68–78 (2017). 37. Ortega-Regules, A., Ros-García, J. M., Bautista-Ortín, A. B., López-Roca, J. M. & Gómez-Plaza, E. Diferences in morphology and composition of skin and pulp cell walls from grapes (Vitis vinifera L.): technological implications. Eur. Food Res. Technol. 227, 223 (2007). 38. Jacob, P., Hirt, H. & Bendahmane, A. Te heat-shock protein/chaperone network and multiple stress resistance. Plant Biotechnol. J. 15, 405–414 (2017). 39. Wang, W., Vinocur, B., Shoseyov, O. & Altman, A. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9, 244–252 (2004). 40. Eckey-Kaltenbach, H., Kiefer, E., Grosskopf, E., Ernst, D. & Sandermann, H. Diferential transcript induction of parsley patho- genesis-related proteins and of a small heat shock protein by ozone and heat shock. Plant Mol. Biol. 33, 343–350 (1997). 41. D’Haese, D., Horemans, N., De Coen, W. & Guisez, Y. Identifcation of late O3-responsive genes in Arabidopsis thaliana by cDNA microarray analysis. Physiol. Plant. 128, 70–79 (2006). 42. Puckette, M. et al. Diferential mRNA translation in Medicago truncatula accessions with contrasting responses to ozone-induced oxidative stress. Mol. Plant 5, 187–204 (2012).

Scientifc Reports | (2021) 11:8114 | https://doi.org/10.1038/s41598-021-87542-y 12 Vol:.(1234567890) www.nature.com/scientificreports/

43. Bohler, S. et al. A DIGE analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism. Proteomics 7, 1584–1599 (2007). 44. Torres, N. L. et al. Gel-based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama. Electrophoresis 28, 4369–4381 (2007). 45. Cho, K. et al. Integrated transcriptomics, proteomics, and metabolomics analyses to survey ozone responses in the leaves of rice seedling. J. Proteome Res. 7, 2980–2998 (2008). 46. Guo, M. et al. Te plant heat stress transcription factors (HSFs): structure, regulation, and function in response to abiotic stresses. Front. Plant Sci. 7, 114 (2016). 47. Rocheta, M., Becker, J. D., Coito, J. L., Carvalho, L. & Amâncio, S. Heat and water stress induce unique transcriptional signatures of heat-shock proteins and transcription factors in grapevine. Funct. Integr. Genomics 14, 135–148 (2014). 48. Pillet, J. et al. VvGOLS1 and VvHsfA2 are involved in the heat stress responses in grapevine berries. Plant Cell Physiol. 53, 1776–1792 (2012). 49. Suzuki, N. et al. Enhanced tolerance to environmental stress in transgenic plants expressing the transcriptional coactivator multiprotein bridging factor 1c. Plant Physiol. 139, 1313–1322 (2005). 50. Carvalho, L. C., Vidigal, P. & Amâncio, S. Oxidative stress homeostasis in grapevine (Vitis vinifera L.). Front. Environ. Sci. 3, 20 (2015). 51. Fichman, Y. & Mittler, R. Rapid systemic signaling during abiotic and biotic stresses: is the ROS wave master of all trades?. Plant J. 102, 887–896 (2020). 52. Conklin, P. L. & Barth, C. Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell Environ. 27, 959–970 (2004). 53. Potters, G., De Gara, L., Asard, H. & Horemans, N. Ascorbate and glutathione: Guardians of the cell cycle, partners in crime?. Plant Physiol. Biochem. 40, 537–548 (2002). 54. Pandey, P., Singh, J., Achary, V. M. M. & Reddy, M. K. Redox homeostasis via gene families of ascorbate-glutathione pathway. Front. Environ. Sci. 3, 1–14 (2015). 55. Carvalho, L. C., Coito, J. L., Colaço, S., Sangiogo, M. & Amâncio, S. Heat stress in grapevine: the pros and cons of acclimation. Plant Cell Environ. 38, 777–789 (2015). 56. Smirnof, N. Vitamin C: Te metabolism and functions of ascorbic acid in plants. in Advances in Botanical Research (eds. Rébeillé, F. & Douce, R.) vol. 59 109–177 (Elsevier Ltd, 2011). 57. Frei, M., Tanaka, J. P., Chen, C. P. & Wissuwa, M. Mechanisms of ozone tolerance in rice: characterization of two QTLs afecting leaf bronzing by gene expression profling and biochemical analyses. J. Exp. Bot. 61, 1405–1417 (2010). 58. Kubo, A., Saji, H., Tanaka, K. & Kondo, N. Expression of Arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide. Plant Mol. Biol. 29, 479–489 (1995). 59. Yoshida, S. et al. Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana. Plant Cell Physiol. 47, 304–308 (2006). 60. Chen, Z. & Gallie, D. R. Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance. Plant Physiol. 138, 1673–1689 (2005). 61. Melino, V. J., Soole, K. L. & Ford, C. M. Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries. BMC Plant Biol. 9, 145 (2009). 62. Fenech, M., Amaya, I., Valpuesta, V. & Botella, M. A. Vitamin C content in fruits: biosynthesis and regulation. Front. Plant Sci. 9, 2006 (2019). 63. Tamaoki, M. et al. Transcriptome analysis of O3-exposed Arabidopsis reveals that multiple signal pathways act mutually antago- nistically to induce gene expression. Plant Mol. Biol. 53, 443–456 (2003). 64. Enami, S., Hofmann, M. R. & Colussi, A. J. Ozone oxidizes glutathione to a sulfonic acid. Chem. Res. Toxicol. 22, 35–40 (2009). 65. Conn, S., Curtin, C., Bézier, A., Franco, C. & Zhang, W. Purifcation, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins. J. Exp. Bot. 59, 3621–3634 (2008). 66. Edge, R., McGarvey, D. J. & Truscott, T. G. Te carotenoids as anti-oxidants—a review. J. Photochem. Photobiol. B 41, 189–200 (1997). 67. Müller-Moulé, P., Conklin, P. L. & Niyogi, K. K. Ascorbate defciency can limit violaxanthin de-epoxidase activity in vivo. Plant Physiol. 128, 970–977 (2002). 68. Latowski, D., Kuczyńska, P. & Strzałka, K. Xanthophyll cycle – a mechanism protecting plants against oxidative stress. Redox Rep. Commun. Free Radic. Res. 16, 78–90 (2013). 69. Havaux, M., Dall’Osto, L. & Bassi, R. Zeaxanthin has enhanced antioxidant capacity with respect to all other xanthophylls in Arabidopsis leaves and functions independent of binding to PSII antennae. Plant Physiol. 145, 1506–1520 (2007). 70. Alboresi, A. et al. Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein. BMC Plant Biol. 11, 62 (2011). 71. Vickers, C. E., Gershenzon, J., Lerdau, M. T. & Loreto, F. A unifed mechanism of action for volatile isoprenoids in plant abiotic stress. Nat. Chem. Biol. 5, 283–291 (2009). 72. Calfapietra, C., Fares, S. & Loreto, F. Volatile organic compounds from Italian vegetation and their interaction with ozone. Environ. Pollut. 157, 1478–1486 (2009). 73. Martin, D. M. et al. Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol. 10, 1–22 (2010). 74. Battilana, J. et al. Functional efect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat favour formation. J. Exp. Bot. 62, 5497–5508 (2011). 75. Velikova, V., Tsonev, T., Pinelli, P., Alessio, G. A. & Loreto, F. Localized ozone fumigation system for studying ozone efects on photosynthesis, respiration, electron transport rate and isoprene emission in feld-grown Mediterranean oak species. Tree Physiol. 25, 1523–1532 (2005). 76. Booker, F. L. & Miller, J. E. Phenylpropanoid metabolism and phenolic composition of soybean [Glycine max (L.) Merr.] leaves following exposure to ozone. J. Exp. Bot. 49, 1191–1202 (1998). 77. Döring, A. S. et al. Deciphering the role of low molecular weight antioxidants in the sensitivity of Melissa ofcinalis L. to realistic ozone concentrations. Ind. Crops Prod. 150, 112369 (2020). 78. Isah, T. Stress and defense responses in plant secondary metabolites production. Biol. Res. 52, 39 (2019). 79. Caretto, S., Linsalata, V., Colella, G., Mita, G. & Lattanzio, V. Carbon fuxes between primary metabolism and phenolic pathway in plant tissues under stress. Int. J. Mol. Sci. 16, 26378–26394 (2015). 80. Rienth, M., Torregrosa, L., Ardisson, M., De Marchi, R. & Romieu, C. Versatile and efcient RNA extraction protocol for grapevine berry tissue, suited for next generation RNA sequencing. Aust. J. Grape Wine Res. 20, 247–254 (2014). 81. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a fexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). 82. Canaguier, A. et al. A new version of the grapevine reference genome assembly (12X.v2) and of its annotation (VCost.v3). Genomics Data 14, 56–62 (2017).

Scientifc Reports | (2021) 11:8114 | https://doi.org/10.1038/s41598-021-87542-y 13 Vol.:(0123456789) www.nature.com/scientificreports/

83. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015). 84. Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015). 85. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). Acknowledgements We would like to thank Marc Farnos for plant management, Sylvain Santoni and Muriel Latreille for RNAseq library preparation. Author contributions A.C., S.S., C.R., K.S.D.L.H., M.R.S., L.T., and G.L.A. designed the experiment. C.R. and L.T. supervised the experiment. L.T. provided the plant material. A.C. and S.S. performed the plant experiment, metabolites and RNA extraction. S.S. carried out transcriptome data analysis. A.C. and S.S. interpreted the results and drafed the manuscript. C.R., A.J.L.J., K.S.D.L.H., M.R.S., L.T., and G.L.A. critically revised the manuscript. All authors read and approved the fnal manuscript.

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